Project Summary One of the most remarkable aspects of biological systems is their ability to operate on many length and time scales to perform their functions. Thus, methods to study those functions need to cover, and preferably connect, such scales. When studying the architecture of cells and their molecular components, imaging modalities range from high-resolution X-ray crystallography and single-particle cryo-EM, where molecules are isolated from their context, to light microscopy and conventional EM, where the localization within the cell is precise, but the structural details are lost. Thus, the grand challenge is to bring together structural and cellular studies. Cryo-electron tomography (CET) holds great promise to bridge together current structural, biochemical, and biophysical approaches. CET allows us to obtain 3D reconstructions of pleiomorphic structures, such as organelles and cells at molecular resolution, providing snapshots of molecular landscapes inside cells captured in action. However, limitations in sample thickness had hindered the application of cryo- EM to eukaryotic samples. We use cryo-FIB milling to micromachine intact cryo-fixed cells into thin-enough regions to study with CET. The resulting data shows readily identifiable features of cells such as individual molecular complexes, proteins embedded in membranes, and filamentous networks, at unprecedented detail. Our data show that we can use this technology to observe the structure of macromolecular complexes deeply embedded and entangled inside the cell, e.g., in the nucleus. Under the right sampling conditions, we can derive the structural dynamics of these macromolecular complexes by statistically comparing individual molecules. Just like super-resolution light microscopy has extended the realm of questions than can be asked in cell biology, I believe that CET will have a transformative impact through its ability to look not only at the location and context, but also at the structure of molecules in their natural environment. The goal of this project is to unleash the full potential of CET by integrating new and available tools around CET, notably, we will: (1) extend the experimental set up to create devices that resemble experimental conditions better than EM grids in order to perturb samples under controlled conditions, (2) use these devices to correlate CET data with light microscopy and powerful tags used in conventional EM, and (3) create computational and modeling tools to analyze such data quantitatively and to integrate data from other sources to reveal the structural dynamics of these complexes and the molecular architectures they form in the cell. Putting in place these techniques will allow us to study structurally uncharted territory: the nuclear periphery. Our first target is the study of the organization of the networks that connect the cytoskeleton and nucleoskeleton formed by LINC complexes, nuclear lamina, and cytoskeletal filaments, in health and disease.